Anti-Citrullinated Protein Antibodies (ACPAs) are specific for Rheumatoid Arthritis (RA) and have been implicated in disease pathogenesis. The fragment antigen-binding domain of ACPA was recently shown to be extensively glycosylated. It is known that glycans play a key role in controlling innate and adaptive immunity, however to date there is limited understanding on the mode of action of glycans in RA. We hypothesise that the glycans on ACPA interact with glycan binding receptors and thus modulate immune responses in RA. Therefore, our aim is to elucidate the glycan effect of ACPA and other glycans on immune cells of RA patients to increase our understanding of RA pathogenesis.Materials and methods
A whole blood flow assay is used to study glycan interactions with leukocytes. Leukocytes were isolated from blood using Ficoll density centrifugation and lysis of erythrocytes. Cells were incubated for 4 hours with 4 µg/ml glycan at 4°C. Used biotinylated glycans include: sialic acid, Lewis-x, mannose, lactosamine, galactosamine and as a negative control glucitol. Glycan binding and identification of immune cell subsets was assessed with flow cytometry using a whole blood flow antibody panel.Results
A whole blood flow assay of four healthy donors showed consistent high binding of mannose to B-cells. Interestingly, no binding of mannose was observed to other immune cells indicating that mannose binds specifically to B cells. At this concentration there was no binding of other glycans to leukocytes. When there is a difference in glycan binding between healthy and RA patients CyTOF3-Helios mass cytometer will be used to have a more in-depth analyse of the interacting leukocyte cell subsets.Conclusion
This study examines the glycan-binding capacity of leukocytes in healthy donors and RA patients via the whole blood flow assay. Our preliminary data indicates specific binding of mannose to B cells. This is an important finding because B cells play a key role in the pathogenesis of RA, as they produce ACPA and are very efficient in antigen presentation. Further studies on glycan binding to other key immune cells in RA may aid in elucidating their role in the pathogenesis of RA.